For the past several decades, assays have been performed using fluorescence, chemiluminescence, or other means of generating a signal in response to an analyte. Currently, many assays are performed by measurement of the intensity of a light signal generated in the total volume of a reaction mixture. The light signal generated can be measured by an optical means, wherein the light signal generated is emitted by a large number of molecules. In a typical embodiment, these assays can be carried out by combining a sample suspected of containing an antigen with a reagent comprising a first antibody attached to a solid support, e.g., a microparticle, to form a reaction mixture. The antigen, if present in the sample, specifically binds to the first antibody. A conjugate, which comprises a second antibody having a label attached thereto, is introduced to the reaction mixture and specifically binds to the antigen, which is specifically bound to the first antibody, which, as stated previously, is attached to the solid support. Such an assay is referred to as a sandwich assay or an immunometric assay. This type of assay is shown schematically in FIG. 1. The signal attributable to the label is then measured after unbound conjugate is removed from the reaction mixture, typically by performing a wash step. The signal that is derived from the total volume of the reaction mixture is measured and then compared to a calibration curve to establish the concentration of antigen present in the sample. When the assay includes a washing step to remove unbound sample analyte before introducing the conjugate antibody, it generally is considered as a “two-step assay”. When the assay introduces the conjugate antibody and the analyte to antibody coated microparticles together without intermediate washing steps, it is considered as “one-step” assay. “Hook effect” or “Prozone phenomenon” is a phenomenon of falsely low values on an assay when an overwhelming amount of antigen is present in a “one-step assay” format. It is caused by insufficient capture antibody and detection antibody in an assay. Such hook effect limits assay dynamic range.
A sandwich assay can detect a wide range of analyte concentrations; typically it can accurately measure analyte concentration by 2-3 orders of magnitude. But it is uncommon to extend analyte detection more broadly, e.g., over 3 orders of magnitude.